Author(s): BARRETT, J., FUNG, H.J.W., LEAVER, M., TINLINE-GOODFELLOW, C.T., ESTAFANOS, S., GILLEN, J.B., BEGGS, A., MOORE, D.R., HODSON, N., Institution: UNIVERSITY OF BIRMINGHAM, Country: UNITED KINGDOM, Abstract-ID: 1303

INTRODUCTION:
Short-term reductions in physical activity occur during illness, injury, or enforced inactivity and are associated with impairments in skeletal muscle metabolism and glycaemic control. Most step-reduction studies in older adults use ≥14-day interventions, leaving early adaptations poorly characterized. Long-read RNA sequencing (LRS) offers enhanced resolution of transcriptional change but its application in inactivity studies is limited. This study investigated skeletal muscle transcriptional and mitochondrial responses after 3-days step-reduction (<1500 steps/day) in older adults.
METHODS:
Twelve individuals (67 ± 4 years; BMI 22.8 ± 2.7 kg·m⁻²) had a skeletal muscle biopsy pre and post step-reduction. Citrate synthase (CS) activity and mitochondrial electron transport chain (ETC) complex I–V protein abundance were assessed by spectrophotometric DTNB-based assay and immunoblotting as markers of mitochondrial content. RNA extraction was subjected to LRS (MinION, Oxford Nanopore Technologies). Gene-level expression was quantified using the bambu pipeline, with paired differential expression analysis using linear models and Benjamini–Hochberg false discovery rate (FDR) correction. Rank-based gene set enrichment analysis was conducted using the fgsea package against KEGG and Reactome databases (FDR<0.05), with group effects examined using linear regression.
RESULTS:
Of 34,039 expressed genes (protein-coding and non-coding transcripts) detected, 24 were differentially expressed (FDR<0.05) after step reduction. Downregulated transcripts included mitochondrial and lipid metabolism genes (FABP3, SLC25A20, CYCS, CKMT2) and mechanosensitive regulators (ANKRD2, CSRP3), while DDIT4L (mTORC1 inhibitor), G0S2 (lipolysis inhibitor), and TMEM70 (complex V stabilizer) were upregulated. Gene set enrichment revealed marked suppression of mitochondrial oxidative metabolism (NES=−3.10, FDR=9.3×10⁻³⁰; KEGG NES=−2.87, FDR=5.7×10⁻²⁰) and mitochondrial translation pathways (NES=−2.80, FDR=1.12×10⁻¹⁵), alongside enrichment of growth factor and second messenger signalling pathways (NES=+1.68, FDR=2.2×10⁻⁵). Analysis by habitual step count (high: 22,660 ± 6,158; low: 9,197 ± 2,016 steps/day) revealed no additional differential expression. CS activity did not change following step-reduction (mean Δ=−17.9 nmol·min⁻¹·mg⁻¹; 95% CI −70.8 to 35.1; p=0.470), nor did the ΔCS differ by habitual step count (p=0.399). Immunoblotting showed a directional reduction in Complex II protein abundance (mean Δ=−0.107 AU; 95% CI −0.190 to −0.038), but was not significant (p=0.095). No other ETC complexes changed, and no transcriptional associations with CS or ETC protein content were observed after adjustment for habitual activity, sex and multiple comparisons.
CONCLUSION:
These findings show that 3 days reduced step count induces rapid, coordinated suppression of skeletal muscle mitochondrial gene programmes in older adults, preceding detectable changes in mitochondrial content and independent of habitual activity levels.